Cancer-Associated Fibroblasts from Invasive Breast Cancer Have an Attenuated Capacity to Secrete Collagens

INTERNATIONAL JOURNAL OF ONCOLOGY 45: 1479-1488, 2014

Cancer-associated fibroblasts from invasive breast cancer have an attenuated capacity to secrete collagens

Zhixuan Fu1,2*, Peiming Song1*, Dongbo Li3, Chenghao Yi1, Huarong Chen1, Shuqin Ruan1, Zhong Shi1, Wenhong Xu1, Xianhua Fu1 and Shu Zheng1

1Key Laboratory of Cancer Prevention and Intervention (China National Ministry of Education), The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310009; 2Department of Surgical Oncology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022; 3Cardiovascular Ward of Geriatric Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China

Received April 22, 2014; Accepted June 18, 2014

DOI: 10.3892/ijo.2014.2562

Abstract. Normal fibroblasts produce extracellular stroma of invasive breast cancer. These studies showed that matrix (ECM) components that form the structural framework although CAFs from invasive breast cancer possess an activated of tissues. Cancer-associated fibroblasts (CAFs) with an activated phenotype, they secreted less collagen and induced less ECM phenotype mainly contribute to ECM deposition and construc- deposition in cancer stroma. In cancer tissue, the remodeling of tion of cancer masses. However, the stroma of breast cancer stromal structure and tumor microenvironment might, therefore, tissues has been shown to be more complicated, and the mecha- be attributed to the biological changes in CAFs including their nisms through which CAFs influence ECM deposition remain protein expression profile. elusive. In this study, we found that the activated fibroblast marker α-smooth muscle actin (α-SMA) was only present in the stroma Introduction of breast cancer tissue, and the CAFs isolated from invasive breast cancer sample remained to be activated and proliferative The main function of fibroblasts is secreting the components in passages. To further assess the difference between CAFs and of extracellular matrix (ECM). Without activation, fibroblasts normal breast fibroblasts (NFs), MALDI TOF/TOF‑MS was are dormant in the ECM, however, once activated, fibroblasts used to analyze the secretory proteins of primary CAFs and could be involved in secreting higher levels of ECM compo- NFs. In total, 2,903 and 3,023 proteins were identified. Mass nents and exert a stronger proliferative effect (1,2). In the 1970s, spectrum quantitative assay and data analysis for extracellular Ryan et al originally described the activation of fibroblasts in proteins indicated that the CAFs produce less collagens and granulation tissues during wound healing progression (3,4). matrix-degrading enzymes compared with NFs. This finding Activated fibroblasts are also referred to as myofibroblasts was confirmed by western blot analysis. Furthermore, we due to their expression of α-smooth muscle actin (α-SMA), discovered that reduced collagen deposition was present in the as well as other important functions (5,6). Carcinoma tissues are mainly composed of tumor cells and stromal cells, the latter include fibroblasts, endothelial and inflammatory cells. Fibroblasts, termed cancer-associated fibro- Correspondence to: Professor Shu Zheng, Key Laboratory of blasts (CAFs) represent the most abundant cellular components Cancer Prevention and Intervention (China National Ministry of in cancer stroma. The heterotypic and multicellular interactions Education), The Second Affiliated Hospital, College of Medicine, among cells, soluble factors, signaling molecules, extracellular Zhejiang University, Hangzhou, Zhejiang 310009, P.R. China matrix construct a new biological system termed the ‘tumor E-mail: [email protected] microenvironment (TME)’. Researchers have demonstrated that tumor cells and the TME coevolve through continuous *Contributed equally paracrine communication (7), which not only creates a dynamic Abbreviations: CAFs, cancer-associated fibroblasts; NFs, normal signaling circuitry that promotes cancer initiation and progress, bread fibroblasts; ECM, extracellular matrix; MAL-DI TOF/TOF-MS, but also induces the activation of fibroblasts (8). CAFs present matrix-assisted laser desorption/ionization time of flight mass in cancer stroma exhibit an activated phenotype analogous to spectrometry; MRM, multiple reaction monitoring; CPS, counts per that of fibroblasts involved in wound healing or fibrosis (9,10). second; SMA, smooth muscle actin; PDGFR, platelet derived growth Indeed, Dvorak originally considered tumors to be ‘wounds factor receptor that do not heal’, because of the similarity with granulation tissue (11). Increasing evidence supports the role of CAFs as Key words: cancer-associated fibroblasts, extracellular matrix, tumor a key regulator of the paracrine signaling required for cancer microenvironment, mass spectrometry, desmoplasia progression (12,13). In contrast to resting fibroblasts, activated 1480 Fu et al: cancer-associated fibroblasts secrete less collagen

CAFs can be identified by their expression of vimentin,α -SMA, penicillin and 100 µg/ml streptomycin; Sigma), ascorbic acid fibroblasts activated protein (FAP), and platelet-derived growth (20 ng/ml), and FGF-basic (10 ng/ml; Sigma). After one week factor receptor (PDGFR-β) (14-16). Kalluri has indicated that of incubation in a humidified incubator with 5% CO2 atmo- up to 80% of stromal fibroblasts in breast cancer display this sphere, tissue debris was removed. Once primary cells reached activated phenotype (17). Furthermore, evidence indicates that 80% confluence, they were harvested and reseeded in a flask. CAFs neither revert back to normal fibroblasts nor undergo elimination via apoptosis (18). Characterization of CAFs/NFs. CAFs and NFs were seeded CAFs are further characterized by their production of into 24-well plates. The culture medium was removed and abundant ECM proteins, which are responsible for the stiffening cells were fixed for 20 min with SafeFix solution (Sinai) appearance of cancer (19,20). The deposition of ECM induced once 70-90% confluence was reached, followed by treating by CAFs in tumor stroma is known as desmoplasia, which has with 0.2% Triton X-100 for 15 min, and incubating in been shown to be extensive in or around tumors, particularly non‑immunone goat serum for 20 min. Rabbit anti-vimentin, in human breast cancer and pancreatic carcinoma (21,22). A anti-FSP-1, anti-fibronectin antibodies and mouse anti- study has shown that the compression of ECM in turn leads to α-SMA, anti‑cytokeratin (pan) antibodies (Maxin) were used the concentration of soluble factors that promote tumorigenesis in incubation at 4˚C as the primary antibody. Anti-mouse and in an autocrine and paracrine manner (13). However, Walker anti-rabbit fluorescent secondary antibodies were then applied has reported that the stroma in a range of primary breast carci- accordingly. After rinsing, cells were counterstained with nomas could vary from being predominantly cellular with little DAPI (Sigma), and images were obtained with a laser scan- collagens to being a dense collagenous stroma with apparently ning confocal microscope. Immunocytochemistry by DAB few stromal cells (23). These results are contradictory to many staining was conducted. The methods for western blot analysis research results on the universal deposition of ECM in cancer are presented in the section ‘Western blot analysis’. tissue. Therefore, it is necessary to elucidate the relationship between activated CAFs and their function in ECM deposition. Cell growth evaluation. Briefly, 1,500 CAFs and NFs were In this study, we aimed to investigate the secretomics of CAFs seeded per well in 96-well plates. Cell viability was assessed by mass spectrometry and other means in order to elucidate the daily by adding 20 µl of 3-(4,5-dimethylthiazol-2-thiazyl)- relationship between CAFs and ECM deposition in breast cancer. 2,5-diphenyl-tetrazolium bromide (MTT; 5 mg/ml in PBS) to each well. MTT was also added to the control wells without Materials and methods cells. Each sample included six replicates. Following 4‑h incubation at 37˚C, the medium was removed. An aliquot of 200 µl Specimens. This study was based on a well-characterized DMSO was transfered to each well, and the plate was agitated series of TNM stage T2-T3 primary invasive breast carcinoma for 15 min. The absorbance at 570 nm was then detected. For cases and fibroadenomas. Invasive breast cancer was selected MTT assays that were performed on the same day with cell for study due to its typical desmoplastic response and the pres- seeding, the cells were allowed to attach for 3 h before the ence of large numbers of activated CAFs. The fresh specimens addition of MTT. Growth curves were constructed by plotting of invasive carcinoma were collected from cancer patients who absorbance (mean ± SD) against time. accepted radical mastectomy at the Second Affiliated Hospital of Zhejiang University. Written consent was obtained from all Extraction of secretory proteins from CAFs and NFs. When patients and this study was approved by the ethics committee CAFs and NFs reached 70-80% confluence, the medium was of Zhejiang University. Tissue specimens used in immunohis- removed, and the flasks were washed gently three times with tochemistry and histochemistry were obtained from the tissue PBS. Cells were then cultured in conditioned medium (CM; bank of the Cancer Institute, Zhejiang University. serum-free, phenol red-free DMEM medium supplemented with ascorbic acid 20 ng/ml). Following 18-h incubation, the Immunohistochemistry (IHC) and histochemistry. In total CM was collected and the cell debris was removed by centrifu- 160 cases of invasive breast cancer, 40 adjacent normal gation for 10 min, 4˚C (200 x g), followed by sterile filtration breast tissue and 6 fibroadenoma specimens were obtained. (0.22 µm; Millipore). Protease inhibitors were applied to After the tissues were fixed with 4% paraformaldehyde and prevent protein degradation. The protein present in the CM paraffin-embedded, sections (5 µm) were prepared, and IHC was concentrated by an ultrafiltration (3,000 Da; Millipore) was performed with monoclonal antibodies against α-SMA and precipitated in acetone overnight at -20˚C. The sediment and vimentin (Maxin). The IHC protocol was described as was then washed twice with acetone, followed by resuspended previously (24). H&E staining and Masson trichrome staining in protein solution (RIPA, Beyotime). The protein concentra- (Maiwei) were performed on the three kinds of specimens. tion was then measured with a standard Bradford protein Images were obtained using laser confocal and optical micro- assay (Bio-Rad). Prior to the collection of condition medium, scopes. β-galactosidase staining was performed to exclude senescent cells. CM samples were collected from three pairs of homolo- Primary culture of normal fibroblasts (NFs) and CAFs.Fresh gous CAFs and NFs. invasive cancer specimens and paired adjacent normal breast tissue samples (>3-5 cm away from the tumor) were collected MALDI TOF/TOF-MS for global screening of secretory under sterile conditions. The specimens were sectioned into protein. In total, 200 µg of secretory proteins from CAFs and approximately 1-mm3 pieces and placed in DMEM/F12 (Gibco) NFs were separated by 12% SDS-PAGE, and the resulting supplemented with 10% FBS (Gibco), antibiotics (100 U/ml gel was stained with Commassie Blue Fast Staining Solution INTERNATIONAL JOURNAL OF ONCOLOGY 45: 1479-1488, 2014 1481

Figure 1. The difference of α-SMA expression in invasive breast cancer, normal breast tissue and breast fibroadenoma. Tissues detected by immunohistochemistry and immunofluorescence showed that:α -SMA expression was widely observed in the stroma of invasive breast cancer tissue [the brown area in (A), DAB staining. (C) Green, α-SMA; red, vimentin, pink arrows]. While, little α-SMA expression was observed in normal breast/fibroadenoma stromal region (B, white arrows), and α-SMA was only present in myoepithelium, vimentin was negative (B and D, pink arrows, no red). (A, B, C and D), 200‑fold.

(Invitrogen). Each lane was then cut into 15 even sections (within 8 passages) were washed twice with PBS, and then and digested as previously reported (25). Briefly, all sections lysed in RIPA and ultrasonicated on ice. The lysates were then were destained and dehydrated, and proteins were reduced centrifuged for 30 min at 4˚C (10,000 x g) and the supernatants with dithiothreitol and alkylated with iodoacetamide (IAA, were collected and stored at -80˚C. The extraction of secretory Sigma). After alkylation, the samples were incubated with proteins was performed as outlined in ‘Extraction of secretory sequencing‑grade trypsin (Promega) at 37˚C for 20 h. The proteins form CAFs and NFs’ section. peptides were then subjected to extraction (50% acetoni- Western blots. Equal amounts of protein samples from trile, 5% formic acid) and lyophilized under vacuum for CAFs and NFs (intracellular or secretory proteins) were MALDI TOF/TOF (Bruker Ultraflextreme) detection. All of separated on a 12% SDS-PAGE gel, transferred to PVDF the peptides were retrieved from the UniProtKB website. membranes (0.2 µm; Millipore), and then incubated with various primary antibodies (α-SMA, PDGFR-β, collagen α-I , Label-free quantitative assay for secretory proteins of interest collagen α-III, diluted 1:2,000 in 5% defatted milk, GADPH, Trypsin digestion. Protein samples from each fraction were diluted 1:5,000) overnight at 4˚C. The membranes were blotted reduced with DTT and alkylated with IAA. After being diluted with HRP conjugated secondary antibodies (Epitomics), in a solution of 100 mM NH4HCO3, the protein mixture was developed using an ECL substrate (Pierce) and exposed to digested by sequencing-grade trypsin at 37˚C for 20 h. Kodak Biomax MR film. Desalting peptides and multiple reaction monitoring (MRM) assay. The tryptic peptide mixture was desalted using Bioinformatics analysis and statistical analysis. MS data were a porous C-18 reversed-phase resin (Pierce) according to the analyzed using Analyst software (version 1.5.1, AB Sciex), manufacturer's instructions. Eluted peptides were lyophi- and sequences were searched in the SwissProt database with lized and redissolved in 10 µl 0.1% FA for 4000 QTRAP® Protein Pilot (version 4.0, AB Sciex). The cellular localization LC/MS/MS System and targeted proteomics assay (MRM of identified proteins was analyzed on the basis of informa- assay model) (26). tion available from UniprotKB. MRM propilot (version 2.1, AB Sciex) and Protein Pilot were used to analyze the amino Western blot analysis acid sequence of trypsin‑digested peptides and determine the Protein extraction. Whole intracellular proteins were extracted optimal peptide sequences for quantitative detection. Statistical in accordance with a standard protocol. Briefly, adherent cells analyses were conducted with SPSS software (version 19.0). 1482 Fu et al: cancer-associated fibroblasts secrete less collagen

Results

Fibroblast activation in breast cancer. To characterize the activation of fibroblasts, the expression of α-SMA was analyzed in invasive breast cancer, normal breast tissue and fibroadenoma. We found the α-SMA positive fibroblasts were widely present in the stroma of invasive breast carcinoma compared to that of normal breast tissues and fibroadenoma (Fig. 1). In normal breast tissues, only the myoepithelial cells distributed along the entire duct-lobular system and the smooth muscle cells around the microvasculature were α-SMA positive, while vimentin was not detected. It has been reported that fully differentiated myoepithelial cells can vanished during cancer progression (27). Therefore, the α-SMA positive cells within the tumor stroma activated CAFs (α-SMA+ vimentin+). A large number of fibroblasts were detected in breast fibroadenoma with abundant ECM, however, these fibroblasts were α-SMA- negative. In summary, our results suggest that activation of fibroblasts only appeared in invasive cancer, which is consistent with findings from previous studies (13). Noteworthy, our data showed that not all the CAFs showed α-SMA expression in the immunofluorescence assay.

CAFs maintain an activated phenotype in vitro. To investigate the characteristics of the primarily cultured cells, we performed immunocytochemistry and immunofluorescence using antibodies against α-SMA, the muscle cell marker desmin, the epithelial cell marker cytokeratin (pan), the mesenchymal cell marker vimentin, the fibroblast marker FSP-1 and fibronectin (Fig. 2). The results indicated that only CAFs were α-SMA-positive, while vimentin and fibronectin could be detected in both CAFs and NFs. Although FSP-1 was expressed in both cell types, relatively few positive cells were observed. Our results demonstrated that cytokeratin was not expressed in either type of cells. It was notable that the CAFs from one specimen showed weak expression of desmin which would be expected to be absent in breast fibroblasts. The expression of α-SMA was confirmed by western blot analysis (Fig. 2I). Furthermore, another activation marker PDGFR-β was detected by western blot analysis (Fig. 2I) but the result showed no difference between CAFs and NFs. These results demonstrated that CAFs and NFs were kindred cell types with the nature of fibroblasts after multiple passages in vitro, CAFs maintained an activated phenotype.

The proliferative capacity of CAFs and NFs. The MTT assays were carried out to analyze the proliferation capability of CAFs and NFs. The results were normalized statistically (Fig. 3A), suggesting that the proliferation ability of CAFs were either stronger than or equal to that of NFs (days 5, 6 and 7, p<0.05 or p>0.05). Figure 2. Characterization of CAFs and NFs activation. 1) Immuno- cytochemistry: The cells were labeled by DAB, fluorescent secondary The detection of secretory proteins from CAFs and NFs. The antibodies (green, red) and DAPI (blue). a) Immunocytochemistry showed CAFs were fractionally α-SMA-positive (A and C), while NF completely 3 pairs of samples of secretory proteins obtained from CM of lacked α-SMA expression (B and D). b) CAFs and NFs both express CAFs and NFs were globally scanned by MALDI TOF/TOF-MS. vimentin (E and F). In (E), CAFs demonstrated weak expression of desmin, Results showed in total 2,903 proteins in CAFs and 3,023 which was absent in NF. c) No expression of cytokeratin (pan) was observed proteins in NFs (data not shown). Among them, 2,811 proteins in CAFs or NFs, while the expression of FSP-1 was found only in a small proportion of CAFs and NFs (G and H). (A and B), 50-fold; (C, D, E, F, G were shared in these two samples. The extracellular proteins and H), 200-fold. 2) Western blot analysis for α-SMA and PDGFR-β assay: included matrix metalloproteinases (MMP1, MMP2, MMP3, CAFs expressed α-SMA, while NF almost completely lacked it. But, there MMP9, MMP10, MMP11 and MMP14), cathepsins, plas- was no expression difference on PDGFR-β between CAFs and NFs (I). INTERNATIONAL JOURNAL OF ONCOLOGY 45: 1479-1488, 2014 1483

Figure 3. Cell growth curve and cartogram of secretory protein expression from CAFs and NFs. 1) Chart A shows the growth curve of CAFs and NFs. Three pairs of homologous CAFs and NFs were evaluated. On days 5, 6 and 7, the data did not provide evidence that the proliferation of CAFs in vitro was weaker than that of NFs (p<0.05 or p>0.05). 2) Chart B is a cartogram of peptide ion intensity ratios (CPSCAFs/ CPSNFs, mean ± SD, n≥3). To delimit the expression difference, ratios ≥1.5 and ≤0.7 were specified as the difference boundary values of protein expression, and ratio =1 was assigned as an equivalent reference line. According to these rules, only two proteins in the selected proteins had higher expression in CAFs (blue), while more proteins showed lower expression (red). Chart B is divided into three parts: part b, matrix-degrading enzymes and protease inhibitors; part c, extracellular matrix components; and part d, other proteins. In most of the ratios parts b and c are lower than 1. These proteins included the EMC ingredient.

minogen activator inhibitors (PAI), cystatins, collagens and proteins generated by CAFs and NFs, we chose to profile other extracellular components. Several membrane proteins ECM-related proteins and other extracellular protein compo- were also identified, including cadherin, integrin and nents, as well as several cytokines (Table I) by MRM, Applied growth factor-binding protein. Regarding collagens, CAFs Biosystems, and MDS Inc). The ion-intensity values (CPS) of and NFs were found to have different expression profiles: representative peptides from different proteins were collected, collagen α-1(III), collagen α-5(VI) were only discovered in and the CPS values of a same ion-peptide (in a same batch

NFs, while collagen α-1(XII) and collagen α-2(V) appeared in testing) were calculated, ion-intensity ratio = CPSCAFs/CPSNFs. CAFs. Four of the six types were detected in CAFs. A ratio of each protein was then obtained from data from the same batch testing. To delimit the expression difference, we Label-free quantitative assay for selected proteins by MRM. specified ratios ≥1.5 and ≤0.7 as the boundaries of protein To quantitatively identify the differences between secretory expression, with ratio =1 as an equivalent reference line. 1484 Fu et al: cancer-associated fibroblasts secrete less collagen

Table I. Selected extracellular proteins and peptides for quantitative assay by mass spectrometry (4000 QTrap LC/MS/MS, multiple-reaction monitoring model).

Protein ID Protein name Abbreviation The sequence of ideal peptides

P02452 Collagen alpha-1(I) chain COL1A1 DGEAGAQGPPGPAGPAGER, ADDANVVR, GPAGPQGPR P02461 Collagen alpha-1(III) chain COL3A1 GPVGPSGPPGK P20908 Collagen alpha-1(V) chain COL5A1 ENPGSWFSEEK P12109 Collagen alpha-1(VI) chain COL6A1 IALVITDGR, LKPYGALVDK, TAEYDVAYGESHLFR, VPSYQALLR Q99715 Collagen alpha-1(XII) chain COL12A1 ALALGALQNIR, VILTPMTAGSR, NSDVEIFAVGVK P08123 Collagen alpha-2(I) chain COL1A2 GEAGAAGPAGPAGPR, TGEVGAVGPPGFAGEK, TGHPGTVGPAGIR P12110 Collagen alpha-2(VI) chain COL6A2 DYDSLAQPGFFDR, LFAVAPNQNLK P12111 Collagen alpha-3(VI) chain COL6A3 qINVGNALEYVSR, QLGTVQQVISER, VGLEHLR P05997 Collagen alpha-2(V) chain COL5A2 SLSSQIETMR Q16363 Laminin subunit alpha-14 LAMA14 AIEHAYQYGGTANSR P11047 Laminin subunit gamma-1 LAMC1 LSAEDLVLEGAGLR, LVGGPMDASVEEEGVR, NTIEETGNLAEQAR P07942 Laminin subunit beta-1 LAMB1 IPSWTGAGFVR P35555 Fibrillin-1 FBN1 YLIESGNEDGFFK P02751 Fibronectin FN1 NLQPASEYTVSLVAIK, NTFAEVTGLSPGVTYYFK, TYHVGEQWQK, VGDTYERPK P14780 Matrix metalloproteinase-9 MMP9 LGLGADVAQVTGALR P03956 Interstitial collagenase MMP1 SQNPVQPIGPQTPK, WEQTHLTYR, VTGKPDAETLK, DGFFYFFHGTR Q14515 SPARC-like protein 1 SPARCL1 LLAGDHPIDLLLR, MRDWLK P08254 Stromelysin-1 MMP3 FLGLEVIGK P50281 Matrix metalloproteinase-14 MMP14 SPQSLSAATAAMQK, AVDSEYPK Q96CG8 Collagen triple helix repeat-containing protein 1 CTHRC1 ESFEESWTPNYK P20742 Pregnancy zone protein PZP ATVLNYLPK, GPTQDFR P01033 Metalloproteinase inhibitor 1 TIMP1 GFQALGDAADIR, SEEFLIAGK P01034 Cystatin-C CST3 ALDFAVGEYNK, LVGGPMDASVEEEGVR P05121 Plasminogen activator inhibitor 1 PAI-1 QVDFSEVER, TPFPDSSTHR P05120 Plasminogen activator inhibitor 2 PAI-2 TPVQMMYLR Q15113 Procollagen C-endopeptidase enhancer 1 PCOLCE GFLLWYSGR P07585 Decorin DCN VSPGAFTPLVK, AHENEITK, DLPPDTTLLDLQNNK P01009 Alpha-1-antitrypsin SERPINA1 QINDYVE P10145 Interleukin 8 TYSKPFHPK P61812 Transforming growth factor-β TGF-β EGVYTVFAPTNEAFR, ILGDPEALR, SPYQLVLQHSR P08253 72 kDa type IV collagenase MMP2 AFQVWSDVTPLR, IIGYTPDLDPETVDDAFAR P02771 Alpha-fetoprotein AFP YIQESQALAK

The expression differences of selected proteins are shown in were found for other proteinase inhibitors between CAFs and Fig. 3B. NFs. When metalloproteinases were tested, MMP1, MMP3 and Our data showed α-1-antitrypsin expression was upregu- MMP14 were found to be downregulated in CAFs compared lated, while metalloproteinase inhibitor 1 expression was with NFs. As for the ECM components, the data demonstrated downregulated in CAFs. No differences in protein expression that the expression levels of collagen α-1(II), collagen α-1(VI), INTERNATIONAL JOURNAL OF ONCOLOGY 45: 1479-1488, 2014 1485

Figure 4. Validation of protein expression differences. (A) The ion intensity difference of representative paired peptides [α-1 antitrypsin, collagen α-1 (VI) and MMP3] from homologous CAFs and NF. (B) The results of western blot analysis with anticollagen I and III antibodies (the bands appeared at approximate 95 and 190 kDa). Equivalent protein concentrations from paired homologous CAFs and NFs were loaded in every lane. GAPDH from paired CAFs, NFs was used as an internal control. (C) The grayscale statistical analysis of western blotting (collagen I and III; error bars, mean ± SD, n=3).

collagen α-2(VI), fibrillin and fibronectin were all lower Reduction of collagen deposition in breast cancer stroma. To in CAFs than in NFs; only procollagen C-endopeptidase further validate the collagen reduction found in the label-free enhancer 1 was highly expressed in CAFs. When ratio = 1 was quantitative assay, we performed HE and masson trichrome set as an equivalent reference line for the analysis, we found staining to assess the collagen density in breast cancer tissue, a broad decrease in expression of secretory proteins in CAFs. normal tissue and breast fibroadenoma. The HE staining sections suggested a significantly reduction of ECM amount Western blot analysis validation of collagen expression. In in cancer stroma or cancer reactive stroma (Fig. 5A-C). order to verify our results, we carried out western blotting Masson trichrome staining exhibited more obvious differ- to verify the collagen expression beyond the threshold (≥1.5 ences in collagen deposition. In order to quantify the results, or ≤0.7). The results showed that there was a significant the density of the blue color in masson trichrome staining was difference in expression of collagens between CAFs and NFs analyzed by ImagePro Plus6.0. Statistical analysis showed (Fig. 4). These results suggest that the setting of the boundary that there were significant differences in collagen deposi- values was rational, and strengthened the reliability of data tion among cancer stroma, paracarcinoma, normal tissue from our label-free quantitative assay. These findings also and fibroadenoma, whereby cancer stroma showed the least provide further evidence to prove the decreased expression of collagen deposition (p=0.000) compared with the other three ECM components in CAFs. kinds of stroma. This assay and analysis further confirmed 1486 Fu et al: cancer-associated fibroblasts secrete less collagen

Figure 5. The deposition of ECM and collagen in tissue. 1) (A and B) H&E staining showing that ECM is significantly reduced in cancer stroma or cancer reactive stroma, with or without increased numbers of stromal cells. (C) H&E staining of normal breast tissue demonstrating a rich ECM and lack of stromal cells (green arrow). 2) (D, E and F) Masson trichrome staining depicting collagen in blue. In (D), the cancer stroma (black arrow) is lighter in blue color compared with paracarcinoma stroma, normal breast stroma (F) and fobroadenoma stroma (green arrow). The tissue of breast fibroadenoma showed the highest density of collagen deposition. 3) (G) Results of statistical analysis for collagen density (the blue area shown by masson trichrome staining. Four areas: cancer stroma, paracancer stroma, normal tissue stroma and fibroadenoma, in D, E and F). Cancer stroma showed the least collagen deposition comparing with the stroma from paracarcinoma, normal tissue and fibroadenoma (error bars represent mean ± SEM, n=20). (A, B and C) 170-fold; (D, E and F) 130-fold.

the collagens reduction observed in the label-free quantita- stay inactivated by constitutively expressing vimentin and tive assay. As collagens are the most abundant proteins of other markers (29). Once activated (by some pathological the ECM and offer structural support for resident cells, the factors, for example, wound healing or fibrosis), they can decrease of collagen deposition implies a reduction of ECM immediately take on enhanced proliferative and ECM gener- in cancer stroma. ating capability and become markers-positive, for example, α-SMA and PGDFR-β. The series of responses finally leads to Discussion wound healing progression such as wound contraction, angio- genesis and stimulation of epithelial cell proliferation (30). Usually, the major function of fibroblasts is maintaining the It has been reported that CAFs in tumor microenvironments structural framework of tissues by continuously secreting also acquire a similar activated phenotype, and function in a ECM components (28). Under normal conditions, fibroblasts co-evolutionary manner with cancer cells (31,32). INTERNATIONAL JOURNAL OF ONCOLOGY 45: 1479-1488, 2014 1487

In the present study, we determined the expression of While fibroblasts with activated traits are expected to α-SMA, which is a major component of the contractile produce more ECM components, evidence in our study apparatus, as a marker of fibroblast activation. While there demonstrated that normal breast stroma and fibroadenoma, are other activation markers that can be used to reflect the rather than invasive breast cancer stroma had more extensive fibroblast activation state, α-SMA was chosen because it is the collagen deposition. The fibroblasts in normal breast stroma and most commonly used. Immunohistochemistry demonstrated fibroadenoma, however, are not α-SMA-positive. The reduc- that CAFs were the only cellular components that expressed tion of collagens within the tumor stroma might be possibly α-SMA in the stroma of invasive breast cancer, but not in caused by an increase in the expression of matrix‑degrading normal and benign tissues (Fig. 1). In agreement, Sappino et al enzymes from tumor cells and tumor stromal fibroblasts, or have found that up to 80% of breast cancers include α-SMA by the decreased secretion of ECM components from CAFs. positive CAFs (33) while evidence from other studies has However, in this study, there was no evidence to suggest that shown absence in breast fibroadenoma (34). Therefore, our there was an increase in the expression of MMPs and other data confirmed that the CAFs found in cancer stroma are in an proteases from CAFs (Fig. 3B). Our study therefore proposes activated state. We also found that after undergoing multiple that the reduction of collagen secretion in CAFs was the major passages (within 8 passages), primary cultured CAFs were still cause of ECM reduction in tumor stroma. able to maintain the expression of activation marker α-SMA In summary, although CAFs from invasive breast cancer (Fig. 2), suggesting that CAFs were able to maintain the acti- obtained the activated phenotype, their capacity of producing vated state in vitro as well as in vivo. ECM components was significantly impaired compared with Some researchers have indicated that activated CAFs with normal fibroblasts and fibroadenoma fibroblasts. In breast PDGFR-β overexpression are responsible for the expansion of cancer, CAFs might have remodeled the stromal structure and tumor stroma and the excessive deposition of ECM (19,22). tumor microenvironment through changes in their biological The increased deposition of ECM in cancer is called desmo- characteristics and the profile of secretory proteins. plasia, which is a process similar to that in organ fibrosis. Usually, the desmoplastic stroma contains increased amounts Acknowledgements of fibrillar collagens and other ECM components (35,36). However, as ECM deposition in breast cancer tissues presents This study was supported by a grant from the National Natural different pathological types (23), further study is required to Science Foundation of China (nos. 83172210 and 30973382). disclose the true relationship between activated CAFs and We thank Dr Shanwei Wang and Dr Jiaping Peng for their ECM deposition. 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